1. Field of Invention
The present invention is directed to provide a recombinant plant expression vector containing isolated cDNA nucleotides sequence encoding sunflower farnesyl pyrophosphate synthase (SFPS) derived from seedlings of Helianthus annus, and also to produce transgenic tobacco plants conferring delayed senescence, greater flower development and increased seed production.
2. Description of the Prior Art
The enzyme farnesyl-pyrophosphate synthase (FPS) catalyzes the synthesis of farnesyl pyrophosphate from isopentenyl pyrophosphate and dimethylallyl pyrophosphate. Farnesyl pyrophosphate (FPP) synthase is a key enzyme in isoprenoid biosynthesis that supplies C15 precursors for the structurally diverse classes of essential metabolites, such as sterols, carotenoids, and prenyl quinones, especially in steroidogenesis and carotenogenesis. The isoprenoid biosynthetic pathway is built around a family of pyrophosphate esters of linear alcohols that contain an increasing number of isoprene units. Beginning with the C5 molecule (DMAPP and IPP), a series of C10 (GPP), C15 (Farnesyl pyrophosphate), C20 (GGPP) and higher molecular weight isoprenoid pyrophosphates are formed by the 1xe2x80x2-4xe2x80x2 addition of isopentenyl pyrophosphate to the growing chain. The 1xe2x80x2-4xe2x80x2 condensation reactions are catalyzed by a family of prenyltransferases that are highly selective for the chain length and a double bond stereochemistry of both substrates and products. FPP is the precursor of the structurally diverse class of sesquiterpenoids such as phytoalexins and lies at a multiple branch point of the isoprenoid pathway, especially in steroidogenesis and carotenogenesis. Several thousands of natural plant products are known to originate from the isoprenoid pathway including chlorophylls, vitamins A, E and K, gibberellines, abscisic acid. Also, previous studies showed that the involvement of FPS in the final steps of natural rubber biosynthesis which includes polymerization of isopentenyl pyrophosphate into rubber. FPS genes have been reported previously in different organisms, including human (Wilkin et al., J. Biol. Chem. 1990 265(8): 4607-4614), yeast (Chambon et al., Curr. Genet. 1990 18(1): 41-46), Arabidopsis thaliana (Cunillera et al., 1996 J. Biol. Chem. 271, 7774-7780), rubber tree (Light et al., J. Biol. Chem. 1989 264(31): 18598-607), and E. coli (Fujisaki et al., J. Bacteriol. 1989 171(10): 5654-5658). Isoprenylation is a posttranslational modification that is believed to be necessary, but not sufficient, for the efficient association of numerous eukaryotic cell proteins to membranes. Additional modifications have been shown to be required for proper intracellular targeting and for the function of certain isoprenylated proteins in mammalian and yeast cells. Attachment of the prenyl groups farnesyl and geranylgeranyl to specific eukaryotic cell proteins by protein prenyltransferases is required for the functioning of a number of cellular processes including signal transduction.
Protein farnesyltransferase (FTase) catalyzes the transfer of the hydrophobic farnesyl group from farnesyl pyrophosphate (FPP) to cellular proteins such as Ras at a cysteine residue near their carboxy-termini. This process is necessary for the subcellular localization of these proteins in the plasma membrane and is required for the transforming activity of oncogenic variants of Ras, making the FTase a prime target for anticancer therapeutics. Farnesylation is required for the membrane partition and function of several proteins, including Ras. Farnesyl-protein transferase inhibitors (FTIs) were developed to prevent Ras processing and for the treatment of cancers harboring mutated Ras. Thus, the inhibitors of protein prenylation have been proposed for the chemotherapy of tumors (Vitale et al., Endocrinology 1999 140(2): 698-704; Kohl et al., Science 1993 260(5116): 1934-1937). Transgenic plants expressing heterologous farnesyl pyrophosphate synthase show two fold increase in seed production, higher chlorophyll contents in leaves and stronger drought-stress tolerance, as compared to non-transgenic or control vector-transformed tobacco plants.
Comparison of the amino acid sequences of FPS ranging from plants to human has shown five distinct regions with high similarity. As a first step to characterize the function of FPS, we have cloned a FPS cDNA and shown that it encodes a functional FPS. The 35S promoter fused to the sunflower SFPS (KCTC 0520BP), which encodes the enzyme farnesyl pyrophosphate synthase, was introduced and the morphological changes were examined in the transgenic tobacco plants (Nicotiana tabacum L.). These constructs allowed the overproduction of farnesyl pyrophosphate (fpp) in tobacco in a random manner. Overproduction of fpp produced a few notable morphological and physiological changes in T0 plant, including darker pigmentation in leaves, new shoot initiation, sustained growth, prolonged and delayed senescence in leaves followed by normal seed settings, more than doubling of flowers, and at least 2-fold increase in seeds production. We tested whether an increased production of the fpp in any organs can influence developmental events, such as growth of axillary shoot meristems, flowering, and seed setting or leaf senescence, in the plant.
Enhanced plant growth and yield has been a major goal of the crop science field. It has been hard to achieve since growth and development is governed by a complex interplay of different genes and their products. One way to increase the productive capacity of biosynthesis would be to apply recombinant DNA technology.
The skills described in this invention will give the agricultural advantages inherent in the transgenic plants as described below. The method this invention includes introducing the sense strand construct of pFPS, regenerating the plant transcribes the nucleotide sequence, and selecting plants which showing dramatic changes in the plant life span, overall seed yield, flower numbers and chlorophyll content. The recombinant expression cassette can be introduced into the plant tissue using Agrobacterium or by a sexual cross.
This invention further provides methods of conferring enhanced growth and development. Although this invention described in conjunction with tobacco plants, these skills may also be used to control growth and development in plants other than tocacco plants.
An object of the present invention concerning the Farnesyl Pyrophosphate Synthase (FPS) of sunflower (Helianthus annus) herein is:
(a) to amplify and sequence sunflower FPS cDNAs by using a pair of universal FPS oligonucleotides probe,
(b) to express FPS cDNAs in bacterial cells and to carry out functional complementation assay in yeast mutant to verify its function,
(c) to generate a line of transgenic tobacco plants to observe the influences of the overexpression of sunflower Farnesyl Pyrophosphate Synthase (SFPS) in vivo,
(d) to produce more numbers of flowers in the plant life time and to produce more than double amounts of seeds in the transgenic plants,
The present invention provides nucleotide and peptide sequences of an isolated cDNA coding for sunflower farnesyl pyrophosphate synthase, a key enzyme for the structurally diverse class of isoprenoid biosynthetic metabolites. The present invention also provides the recombinant plant expression vector comprising said nucleotide sequences and for the host cell into which said DNA sequence in the recombinant plant expression vector has been introduced to produce transgenic tobacco plants.